Bioactive PKS–NRPS Alkaloids from the Plant-Derived Endophytic Fungus Xylaria arbuscula

A novel hybrid PKS–NRPS alkaloid, xylarialoid A (1), containing a 13-membered macrocyclic moiety and [5,5,6] fused tricarbocyclic rings, together with ten known cytochalasins (2–11), was isolated from a plant-derived endophytic fungus, Xylaria arbuscula. The chemical structures of all compounds were elucidated using 1D and 2D NMR, HR ESIMS spectroscopic analyses, and electronic circular dichroism (ECD) calculation. Compounds 1–3 and 10 exhibited significant antitumor activities against A549 and Hep G2 cell lines, with IC50 values of 3.6–19.6 μM. In addition, compound 1 showed potent anti-inflammatory activity against LPS-induced nitric oxide (NO) production in macrophage RAW 264.7 cells (IC50, 6.6 μM).


Introduction
The Xylaria genus belongs to the family of Xylariaceae, which is commonly found among saprophytic and endophytic fungi [1]. Several studies have proved that the genus Xylaria can produce a variety of bioactive secondary metabolites including terpenes, cytochalasins, alkaloids, polyketides, etc. [2,3]. Many of these possess promising biological activities associated with drug discovery, such as cytotoxicity and antimalarial and antimicrobial activities [4]. In the past ten years, a class of polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) alkaloids has attracted the attention of scientists due to their unique structure. These alkaloids have 12-or 13-membered ring systems, γ-lactams, and fused [6,5,6] tricarbocyclic or [6,5,6,5] tetracarbocyclic cores, which were isolated from different fungal species and showed antitumor, antifungal, and antituberculosis biological activities [5][6][7][8][9][10]. The biosynthesis pathway of the PKS-NRPS hybrid alkaloids was through P450-catalyzed cyclization to form paracyclophane, and all of the ring system was constructed from a PK-NRP product via oxidation [11]. Cytochalasins are a class of PKS-NRPS hybrids and have been isolated from the fungi of Chaetomium, Xylaria, Rosellinia, and Zygosporium [12][13][14][15]. They have tricyclic core structures, which are in turn each composed of a perhydroisoindole moiety and a macrocyclic ring. Some of the cytochalasins are phytotoxins or virulence factors, so they possess cytotoxic activities [16][17][18]. In addition, cytochalasins are biosynthesized through a hybrid PKS-NRPS pathway in which Knoevenagel condensation is a key step during the biosynthesis [19]. During our continuous studies on antitumor secondary metabolites from endophytic fungi, we screened a fungus, continuous studies on antitumor secondary metabolites from endophytic fungi, we screened a fungus, Xylaria arbuscula, isolated from the medicinal plant Rauvolfia vomitoria by using LC-MS analysis and bioactivity-guided methods. Its EtOAc extract was subjected to repeated chromatographic purification to afford a novel hybrid PKS-NRPS alkaloid, named xylarialoid A (1), and ten cytochalasins (2)(3)(4)(5)(6)(7)(8)(9)(10)(11) (Figure 1). All eleven compounds were evaluated for their cytotoxic activities against A549 and Hep G2 cell lines and their inhibitory activities toward LPS-induced NO production in macrophages. Herein, we report the isolation and structural elucidation of compound 1 and the antiinflammatory and cytotoxic activities of compounds 1-11.   Figure S1) spectrum indicated the presence of a NH proton signal at δH 8.30 (d, J = 2.2 Hz, 1H); eight olefinic/aromatic protons, including the aromatic proton signals at δH 7.27 (dd, J = 8.3, 2.1 Hz), 6.99 (dd, J = 8.2, 2.5 Hz), 6.90 (d, J = 2.2 Hz), and 6.92 (dd, J = 8.5, 2.0 Hz); a group of terminal olefin protons; and two methyl groups at δH 0.92 (d, J = 6.6 Hz) and 1.01 (d, J = 6.4 Hz). The 13 C NMR and HSQC spectra (Table 1 and Figures S2 and S3) of 1 showed the presence of a 1,4-disubstituted benzene; a disubstituted double bond; a terminal olefin; two methyls; three methylenes; eight methines, including two oxygenated methines; two carbonyl carbons; and an oxygenated quaternary carbon. The planar structure of compound 1 was deduced by HMBC and 1 H-1 H COSY spectra (Figures 2 and S4-S6). The HMBC correlations from H-2 to C-1, C-3, and C-15, from H-3 to C-5 and C-14, from H-6 to C-4, C-8, C-12, and C-13, from H-8 to C-10 and C-12, and from H-13 to C-6 and C-7, combined with the observed 1 19, and H-11/H3-20, indicated the presence of a decahydrofluorene moiety attached to a vinyl group at C-3 and two methyls at C-9 and C-11. The HMBC correlations from NH to C-1′, C-2′, and C-17 and from H-1′ to C-2′ and C-18 suggested the presence of a γ-lactam system. Additional 1 H-1 H COSY correlations of H-5′/H-6′ and H-8′/H-9′ and key HMBC correlations from H2-3′ to C-2′, C-4′, C-5′, and C-9′ and from H-1′ to C-3′ revealed the linkage between a para-substituted phenethyl moiety and a γ-lactam ring. Further HMBC correlations from H-1′ to C-17, C-18, and C-2′, as well as the chemical shifts of C-17, C-1′, and C-2′, indicated the presence of an epoxide moiety at C-1′   Figure S1) spectrum indicated the presence of a NH proton signal at δ H 8.30 (d, J = 2.2 Hz, 1H); eight olefinic/aromatic protons, including the aromatic proton signals at δ H 7.27 (dd, J = 8.3, 2.1 Hz), 6.99 (dd, J = 8.2, 2.5 Hz), 6.90 (d, J = 2.2 Hz), and 6.92 (dd, J = 8.5, 2.0 Hz); a group of terminal olefin protons; and two methyl groups at δ H 0.92 (d, J = 6.6 Hz) and 1.01 (d, J = 6.4 Hz). The 13 C NMR and HSQC spectra (Table 1 and Figures S2 and S3) of 1 showed the presence of a 1,4-disubstituted benzene; a disubstituted double bond; a terminal olefin; two methyls; three methylenes; eight methines, including two oxygenated methines; two carbonyl carbons; and an oxygenated quaternary carbon. The planar structure of compound 1 was deduced by HMBC and 1 H-1 H COSY spectra ( Figure 2 and Figures S4-S6). The HMBC correlations from H-2 to C-1, C-3, and C-15, from H-3 to C-5 and C-14, from H-6 to C-4, C-8, C-12, and C-13, from H-8 to C-10 and C-12, and from H-13 to C-6 and C-7, combined with the observed , and H-11/H 3 -20, indicated the presence of a decahydrofluorene moiety attached to a vinyl group at C-3 and two methyls at C-9 and C-11. The HMBC correlations from NH to C-1 , C-2 , and C-17 and from H-1 to C-2 and C-18 suggested the presence of a γ-lactam system. Additional 1 H-1 H COSY correlations of H-5 /H-6 and H-8 /H-9 and key HMBC correlations from H 2 -3 to C-2 , C-4 , C-5 , and C-9 and from H-1 to C-3 revealed the linkage between a para-substituted phenethyl moiety and a γ-lactam ring. Further HMBC correlations from H-1 to C-17, C-18, and C-2 , as well as the chemical shifts of C-17, C-1 , and C-2 , indicated the presence of an epoxide moiety at C-1 and C-17 in the γ-lactam moiety and a hydroxy at C-2 . Moreover, the HMBC corrections from H-3 and H-15 to C-16 showed that the γ-lactam and decahydrofluorene system were connected through a ketone carbonyl group. Finally, the HMBC correlation from H-13 to C-7 confirmed the presence of a 13-membered ring and completed the whole planar structure of 1. and C-17 in the γ-lactam moiety and a hydroxy at C-2′. Moreover, the HMBC corrections from H-3 and H-15 to C-16 showed that the γ-lactam and decahydrofluorene system were connected through a ketone carbonyl group. Finally, the HMBC correlation from H-13 to C-7′ confirmed the presence of a 13-membered ring and completed the whole planar structure of 1.   The relative configuration of 1 was determined by the ROESY spectrum ( Figure S7) and the coupling constants in the 1 H NMR (Figure 2). The observed ROESY cross-peaks of H-7 with H-14 and H-14 with H-3 suggested that they were cofacial and on the β-orientation. However, the vicinal coupling constant of J 9,10α = 12.2 Hz and the NOE correlations of H-10α with H 3 -9/H 3 -11 revealed that these protons were on the same side, and they were assigned to be α-oriented. The ROESY cross-peaks of H-13 with H-6, H-12, H-15, and H-6 ; H-15 with H-6; and H-1 with H-5 and the amide group (NH) indicated that they were on the α-orientation and that the benzene ring was rotation restricted. Additionally, 1D NOE difference experiments ( Figure S8) were performed to identify the important spatial interactions of 1. The signals of H-13 and H-6 were enhanced when H-15 at δ H 2.37 was irradiated. Furthermore, the coupling constant between H-12 and H-13 (J 12,13 = 8.5 Hz) confirmed their trans-configuration, and H-13 was axial. An additional coupling constant (J = 9.8 Hz) between H-4 and H-5 assigned the ∆ 4,5 geometry as Z. The configurations of the fused [5,6,6] tricarbocyclic ring was similar to that of trichobamide A [20]. In order to determine the absolute configuration of 1, the time-dependent density functional theory (TDDFT) was used to calculate the theoretical electronic circular dichroism (ECD) spectrum (Figure 3). At the B3LYP level, the enantiomer configurations of 1a and 1b were compared by ECD calculation. The experimental ECD curve of 1 was in consistent with the calculated curve of 1a (Figure 3). Therefore, the absolute structure of 1 was finally established as 3S, 6R, 7S, 9R, 11S, 12R, 13S, 14S, 15R, 17R, 1 R, 2 R, and 1 was named xylarialoid A.

Results and Discussion
The relative configuration of 1 was determined by the ROESY spectrum ( Figure S7) and the coupling constants in the 1 H NMR (Figure 2). The observed ROESY cross-peaks of H-7 with H-14 and H-14 with H-3 suggested that they were cofacial and on the β-orientation. However, the vicinal coupling constant of J9,10α = 12.2 Hz and the NOE correlations of H-10α with H3-9/H3-11 revealed that these protons were on the same side, and they were assigned to be α-oriented. The ROESY cross-peaks of H-13 with H-6, H-12, H-15, and H-6′; H-15 with H-6; and H-1′ with H-5′ and the amide group (NH) indicated that they were on the α-orientation and that the benzene ring was rotation restricted. Additionally, 1D NOE difference experiments ( Figure S8) were performed to identify the important spatial interactions of 1. The signals of H-13 and H-6 were enhanced when H-15 at δH 2.37 was irradiated. Furthermore, the coupling constant between H-12 and H-13 (J12,13 = 8.5 Hz) confirmed their trans-configuration, and H-13 was axial. An additional coupling constant (J = 9.8 Hz) between H-4 and H-5 assigned the Δ 4,5 geometry as Z. The configurations of the fused [5,6,6] tricarbocyclic ring was similar to that of trichobamide A [20]. In order to determine the absolute configuration of 1, the time-dependent density functional theory (TDDFT) was used to calculate the theoretical electronic circular dichroism (ECD) spectrum (Figure 3). At the B3LYP level, the enantiomer configurations of 1a and 1b were compared by ECD calculation. The experimental ECD curve of 1 was in consistent with the calculated curve of 1a ( Figure 3). Therefore, the absolute structure of 1 was finally established as 3S, 6R, 7S, 9R, 11S, 12R, 13S, 14S, 15R, 17R, 1′R, 2′R, and 1 was named xylarialoid A.

Cytotoxic and Anti-Inflammatory Activities
The cytotoxic activities of compounds 1-11 against A549 and HepG2 were evaluated using the MTT method (Table 2). Compounds 1-3 and 10 exhibited potent cytotoxicity against the A549 and Hep G2 cell lines, with IC50 values between 3.6 and 19.6 μM. Additionally, the anti-inflammatory effects of compounds 1-11 were explored against NO production in LPS-induced RAW264.7 macrophage cells. Among the test compounds, only compound 1 showed the potent inhibitory activity, with an IC50 value of 6.6 μM. The cytochalasins did not display anti-inflammatory activity (≥50 μM).

Cytotoxic and Anti-Inflammatory Activities
The cytotoxic activities of compounds 1-11 against A549 and HepG2 were evaluated using the MTT method (Table 2). Compounds 1-3 and 10 exhibited potent cytotoxicity against the A549 and Hep G2 cell lines, with IC 50 values between 3.6 and 19.6 µM. Additionally, the anti-inflammatory effects of compounds 1-11 were explored against NO production in LPS-induced RAW264.7 macrophage cells. Among the test compounds, only compound 1 showed the potent inhibitory activity, with an IC50 value of 6.6 µM. The cytochalasins did not display anti-inflammatory activity (≥50 µM).

Fungal Material
The fungal strain was isolated from the leaves of the plant Rauvolfia vomitoria belonging to the apocynaceae family, which was collected from Yunnan Province, China, in July 2013. The plant species was identified by Dr. Yi Sun. The fungus was identified as Xylaria arbuscula by analyses of its rRNA gene sequences (GenBank accession number KY951913.1, see Supplementary Materials). The strain, named cuiluo-leaf-1p-e, is currently deposited in the institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences.

Fermentation and Extraction
The fungal strain was cultured on a plate of potato dextrose agar (PDA) at 27.0 ± 0.5 • C for 3 days, which was then inoculated into 110 × 0.5 L Erlenmeyer flasks each containing 40 g sterilized rice and 60 mL water (sterilization at 121 • C for 20 min).
After incubation at 28 ± 0.5 • C for 9 days, the fermented rice media was exhaustively extracted with ethyl acetate by ultrasonication three times, and the organic solvent was evaporated to dryness under reduced pressure to obtain the crude extract. Then, the EtOAc layer was dissolved in methanol and extracted with petroleum ether (1:1) to remove grease from the rice. After extraction, 5.0 g of crude extract was yielded.

Computation of ECD
A conformational search was carried out in the MMFF94 molecular mechanics force field using the MOE software package, and all the conformers within an energy window of 10 kcal/mol were regarded as the initial conformations. The geometry optimization and frequency calculations were performed with Gaussian16 RevB.01, using the ωB97XD or B3LYP functional at the 6-311G(d,p) level of theory to verify the stability and obtain the energies at 298.15 K and 1 atm pressure. The Boltzmann distribution was calculated according to Gibbs free energies of the conformations. ECD calculations were conducted by using the Cam-B3LYP functional at the TZVP level of theory. The Solvation Model Based on Density (SMD) was used as the solvation model. The Boltzmann-averaged ECD spectra were obtained by using SpecDis 1.71 software [21].

Cytotoxicity Assay
The cytotoxic activities of 1-11 against human lung cancer cells A549 and human hepatocellular carcinoma cells HepG2 were determined by the 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2-H-tetrazolium bromide (MTT) assay [22,23]. The cells were cultured in RPMI-1640 containing 10% (v/v) fetal bovine serum (FBS) and 0.4% (v/v) penicillin-streptomycin solution (10,000 units/mL penicillin and 10,000 µg/mL streptomycin, 100×) at 37 • C under 5% CO 2 . The cells were digested by trypsinization and then diluted to a concentration of 1 × 10 4 cells/mL. The diluted cell suspensions were then added into 96-well microtiter plates (200 µL per well) and incubated in a CO 2 incubator at 37 • C for 24 h. After 24 h, 2 µL of test samples was added to each plate well and incubated for 72 h. Adriamycin was used as the positive control, and the blank control contained 2 µL CH 3 OH. After incubation, MTT solution was added, and the plates were incubated for 4 h. The supernatant liquid was removed, and the cells were disrupted with 150 µL of DMSO for 10 min. The absorption was measured at 570 nm.

Anti-Inflammatory Assay
The anti-inflammatory activities of compounds 1-11 were evaluated on lipopolysaccha ride-induced murine macrophage cell lines (RAW 264.7) [24,25]. RAW 264.7 cells were cultured in DMEM high glucose containing 10% FBS at 37 • C under 5% CO 2 until the cells were confluent. For experiment, 2-5 generations of cultured cells were used. The procedure was as follows. First, raw 264.7 cells in logarithmic growth phase were inoculated into 24-well plates at a density of 2 × 10 5 cells·ml −1 (1 mL per well) and incubated in a CO 2 incubator at 37 • C for 24 h. After 24 h, RAW 264.7 cells were induced to inflammation using 1 mg/mL lipopolysaccharide. Second, the Griess method was used to detect the release of NO in cell supernatant. The diluted cell suspensions were added to 96-well plates. To each well, 0, 0.5, 1, 1.5, 2, 4, 6, and 8 µL of NaNO 2 standard solution (1 mol/L) were successively added. To the 96-well plates was added 100 µL of the obtained supernatant. The 96-well microtiter plates were shaken, 100 µL Griess reagent was added into each well, and the absorption was measured at 570 nm. Resveratrol was used as the positive control; DMSO was used as the blank control.